HIV is a complex animal virus

HIV is a complex animal virus

AIDS

The animal virus HIV infects certain key cells of the immune system, destroying the ability of the body to defend itself from  cancer and disease.
The HIV infection cycle is typically a lytic cycle, in which the HIV RNA first directs the production of a corresponding DNA, and this DNA then directs the production of progeny virus particles. The Future of HIV Treatment.
Combination therapies and chemokines offer promising avenues of AIDS therapy.

What is Disease Viruses ? Who are they?

What is Disease Viruses ? Who are they?

Humans have known and feared diseases caused by viruses for thousands of years. Among the diseases that viruses cause are influenza, smallpox, infectious hepatitis, yellow fever, polio, rabies, and AIDS, as well as many other diseases not as well known. In addition, viruses have been implicated in some cancers and leukemias. For many autoimmune diseases, such as multiple sclerosis and rheumatoid arthritis, and for diabetes, specific viruses have been found associated with certain cases.

In view of their effects, it is easy to see why the late Sir Peter Medawar, Nobel laureate in Physiology or Medicine, wrote, “A virus is a piece of bad news wrapped in protein.”
Viruses not only cause many human diseases, but also cause major losses in agriculture, forestry, and in the productivity of natural ecosystems.

Taq DNA polymerase

Taq DNA polymerase:-


  Taq DNA polymerase is a DNA dependent DNA polymerase, first isolated from the hot spring bacterium, Thermus aquatics in 1976 and in 1989. Due to its wide use in molecular biology (primarily PCR), it is termed as ‘Molecule of the Year’. This thermophilic DNA polymerase encodes an 832-amino acid, 94 kDa protein, which consists of two domains.

1. -NH2 domain:  Similar to 5’-3’ exonuclease domain of members of polymerase I family of DNA polymerase

2. -C terminal domain contains a catalytically inactive 3’-5’ exonuclease and a polymerase sub domain, similar to klenow of DNA polymerase I.

The thermal stability of Taq DNA polymerase is attributed to its hydrophobic core and stable electrostatic interactions and high density of proline residues on the surface of the enzyme.

The optimal activity is at 75-800C temperature and at 600C, the activity is reduced by a factor of 2 and at 370C, its activity is reduced to only 10%.

To initiate DNA synthesis, like other DNA polymerases, it also requires a primer that is annealed to the template strand and caries an extensible 3’-OH group.

Taq DNA polymerase requires Mg2+ for its optimal activity. Phosphate buffers inhibit Taq DNA polymerase and therefore should be avoided. The reaction is usually carried out in the presence of Tris buffer at pH 8.3. Because of the lack of a proofreading function, the rate of misincorporation of dNTPs is high in PCR reactions which are catalyzed by Taq polymerase (or any other DNA polymerase that does not have editing domains).
Several mutant forms of native polymerases, are also available like Pfu And vent. Both of them have proofreading activities contributed by 3’-5’ exonuclease.

 Pfu polymerase is therefore known to generate lowest errors while vent is probably intermediate between Taq and Pfu.

 Pfu polymerase is isolated from Pyrococus furiosus Vent is isolated from Thermococcus litoralis (also known as Tli polymerase) 

T7 DNA Polymerase

T7 DNA Polymerase



 The T7 DNA polymerase from T7 bacteriophage has 3’-5’ exonuclease and DNA polymerase activity but lacks 5’-3’ exonuclease domain, which is similar to T4 DNA polymerase. The processivity of this enzyme is quite good that is, the average length of DNA synthesized before the enzyme dissociates from the template, is considerably greater than for other enzymes. Thus, the average length of DNA synthesized by a single molecule of bacteriophage T7 polymerase is much greater than that of DNAs synthesized by other DNA polymerases. The binding and polymerization domain is occupied by the carboxy terminus while the potent 3’-5’exonuclease activity resides on the amino terminus.

The exonuclease activity is completed inactivated by incubating the enzyme with a reducing agent, molecular oxygen, and low concentrations of ferrous ions, for several days. Over 99% of the exonuclease activity is abolished without affecting the polymerization activity by these agents, which cause mutations and site specific modifications. The resulting chemically modified enzyme is marketed under the trade name Sequenase is ideal for determining the sequence of long tracts of DNA by the dideoxy mediated chain termination method. 

Insertion Vectors

Insertion Vectors

Vectors that have a single target site for insertion of foreign DNA are known as insertion vectors. 20% DNA that is not required for lytic growth is removed and therefore insertion of foreign DNA resumes the size back to something like its full length and can be packaged in vitro. Maximum size of DNA that can be accommodated varies from 9- 11 kb. For DNA of larger sizes, high capacity vectors are designed like:-

Vector                size.                     OrI                            Host
Cosmid.            30-45kb.           Col E1                           E.coli
BAC.                 120-300kb.          Replicon forgein      E.coli  
YAC                   250-400kb           ARS                            Yeast

PCR Mediated Gene Cloning

PCR Mediated Gene Cloning

 There are three strategies for cloning PCR products-

 1) T/A cloning is the easiest cloning method. T/A cloning takes advantage of the terminal transferase activity of Taq polymerase and other non-proofreading DNA polymerases which adds a single 3'-A overhang to each end of the PCR product. The resulting PCR product is then ligated into a linear vector with a 3´ terminal 'T' or 'U' at both ends.

 2) Directional cloning. A restriction enzyme target site is introduced into each of the PCR primers. The resulting PCR product and cloning vector are digested with the restriction enzymes to generate complementary ends at the PCR product and the vector which are then ligated.

  3) Blunt-end cloning-Blunt-end PCR product generated by proof-reading polymerase such as the Pfu DNA Polymerase can also be cloned into a blunt-end vector.

The cloning of PCR-amplified fragments into a linear vector is typically a rapid and efficient process. However, not all PCR fragments will clone with the same efficiency into the same vector. These differences may be due to fragment size, insert toxicity, and the complexity of the insert. The size of the fragment being cloned is a primary contributor to the overall cloning efficiency. Large fragments of DNA (≥ 5 kb) are amenable to cloning in high-copy number vectors, yet at a much lower efficiency. Optimization of molar concentration ratios of the vector to insert is critical to ensure efficient cloning. Successful cloning ratios may range from 1:1 to 1:10. For example, if the vector is 3 kb and the insert is 1 kb, one-third the amount of insert needs to be added to attain a 1:1 molar ratio.  

what is Terminal Deoxy Nucleotidyl Transferase?

what is Terminal Deoxy Nucleotidyl Transferase ?

Terminal transferase is an unusual DNA polymerase found only in prelymphocytes and in early staqes of lymphoid differentiation. Synthesis of single stranded tails at the 3’ ends of either single stranded DNA or double stranded DNA with protruding 3’ termini, by the enzyme Terminal Deoxy Nucleotidyl Transferase, is called tailing, can be used to generate protruding ends of defined sequence to facilitate cloning of fragments. It can be used to generate protruding ends of defined sequence, e.g poly A tails on the 3’ ends of the DNA insert and poly T tails on 3’ ends of the vector. Thus, the protruding ends of the DNA insert and vector will base pair under appropriate annealing conditions. Mg2+ cation is preferred when the nucleotide to be added is a purine while Co2+ is preferred for the addition of pyrimidines. The enzyme strongly prefers DNA with protruding 3’ terminus, although blunt or recessed 3’ termini are also used, but less efficiently, in buffers of low ionic strength with Co2+, Mg2+ or Mn2+ as bivalent cations. As many as thousands of deoxynucleotides can be incorporated using this enzyme on a template of DNA. Single nucleotide can be added to the 3’ termini of DNA if modified bases like dideoxynucleotides or cordycepin triphosphates are used instead of natural deoxynucleotide triphosphates.

What is Follicle in Human fertilization? Development if follicle.

What is Follicle in  Human fertilization? Development if follicle.

Follicle cells (from sex cords) surround the Primary oocyte.

The Follicle is the oocyteplus follicle cells

Primordial follicle -follicle cellspartially surround oocyte

Primary follicle –follicle cells form a complete layer Follicle cells form gap junctions with the oocyteand produce Meiotic inhibitory factor.
Follicle cells are called granulosacells

Granulosacell  layer enclosed by the membranagranulosa, a basement membrane that acts as a barrier to capillaries.

Zona pellucidasecreted by oocyteand
follicle cells –with microvillarconnections between the two. 

Ovary cells form 2 more layers –theca interna, theca external.

Follicle Development

Secondary follicle –formation of the antrum(cavity) fluid filled, liquor folliculi Hormone production, androgens and estrogen.

Tertiary or Graffianfollicle–12 hours prior to ovulation.
cumulus oophorus= mound of cells that house the secondary oocyte.

Oogenesiscontrolled by cycles (Menstrual) of hormone release: Hypothalamus to gonadotropinreleasing hormone(GnRH) Anterior to  pituitary Gonadotropins, includesluteinizing hormone(LH)
and Follicle stimulating hormone(FSH)

Ovulation -tertiary follicle protrudes like a blister on the surface of the ovary –then bursts in response to LH and FSH.

Corpus Luteum–Follicle after ovulation –hormone producing Progessterone.

What is Bacteriophage Artificial Chromosomes (BACs) ?

What is Bacteriophage Artificial Chromosomes (BACs) ?

These plasmids are circular DNA molecules carrying conventional antibiotic resistance marker, origin of replication derived from the F factor of E.coli, an ATP driven helicase (repE) to facilitate DNA replication and three loci (parA, parB and parC) for proper partitioning of the plasmid to daughter cells. BAC vectors have no packing constraints and there is no fixed limit to the size of genomic DNA that they accept. Usually the size of DNA is approximately 120-kilo base pairs.  

Yeast Artificial Chromosome (YACs)

Yeast Artificial Chromosome (YACs)

These are linear DNA molecules similar to yeast chromosome. Recombinant YACs are made by ligating large fragments of genomic DNA and then the resultant plasmid is introduced into yeast by transformation. The vector carries selection marker, DNA sequences called as telomere, so that the product can be stabilized inside the yeast cell, an origin of replication called autonomous replication origin, ARS. Large size of DNA can be inserted into YAC vectors, usually between 250kilobases to 400kilobasepairs. Large size of mammalian genomic libraries is also made with approximately 1 megabasepairs of foreign inserts. Insertion of foreign DNA into the cloning site inactivates a mutant expressed in vector DNA and formation of red rather than white colonies by yeast strain is observed. Thus transformants are identified as red colonies, which grow in yeast that is mutant for TRP1 and URA3, which ensure that the cell has received an artificial chromosome and with both the telomeres since it is complimented for both the mutations. And the colony also contains foreign DNA because it is red in color.

Brief note about Ti-plasmid

Brief note about  Ti plasmid

=>
Ti plasmid is a large mega plasmid conjugative plasmid of ~200kb. pTi is lost when Agrobacterium is grown above 28oC, such cured bacteria do not induce crown galls that is, they become a virulent. pTi and pRi, although do not share sequence homology but are unique in following respects:- a) They contain some genes, which are located within their T-DNA which has regulatory sequences recognized by plant cells, while their remaining genes have prokaryotic regulatory sequences. As a result, the former are expressed only in plant cells but not in the Agrobacterium, while the latter are only expressed in the bacterium. b) These plasmids naturally transfer a part of their DNA, called as T-DNA, into host plant cells. The T-DNA usually contains following important functional regions.
1. T-DNA contains oncogenes and opine synthesis genes and is transferred into host plant.
2. Vir region which regulates the transfer of T-DNA
3. Opine catabolism genes for utilization of opines.
4. Origin of replication for propagation in Agrobacterium. The T-DNA contains a 24bp direct repeat border sequence and contains the genes necessary for tumor / possess gene for auxin and cytokinin biosynthesis. All the genes present in TDNA have eukaryotic regulatory sequences. As a result, these genes are expressed only in plant cells but never express in Agrobacterium. The vir region mediates the transfer of TDNA into plant genomes and hence is essential for virulence. The genes of vir region are not transferred but induce the transfer of T-DNA. Also, the genes present in T-DNA are not responsible for its transfer, but the 24 bp direct repeat at both the left and right ends of TDNA is essential for the transfer. The exact mechanism of transfer of T-DNA is not known clearly known but is brought by the vir region. The phenols produced by wounded plant tissue initiates the transfer process. The T-DNA is transferred into the plant cells as single stranded DNA, which increases the efficiency of its transformation. But, as soon as it enters into the plant cell, it is immediately converted into a double stranded form. This form integrates at random sites in the host plant genome by a phenomenon called illegitimate recombination, which are due to sequence of homology in short segments of the host DNA. This integration is usually in low copy numbers. Few vectors are derived from pTi (wild type) due to some problems posed by wild type plasmid eg. The presence of oncogenes causes a disorganized growth, their large size and lack of cloning sites within the T-DNA, which are needed for the insertion of DNA segments that has to be cloned. 

What is electrophoresis buffer ?

What is Electrophoresis buffer?

Several different buffers have been recommended for electrophoresis of DNA. The most commonly used for duplex DNA are TAE (Tris-acetateEDTA) and TBE (Tris-borate-EDTA). DNA fragments will migrate at somewhat different rates in these two buffers due to differences in ionic strength. Buffers not only establish a pH, but provide ions to support conductivity. If water is used instead of buffer there will be no migration of DNA in the gel and conversely, if concentrated buffer is used like a 10X solution instead of 1X, heat will be generated in the gel which is enough to melt it. 

What is Ethidium bromide?

What is Ethidium bromide?

Answer- Ethidium bromide is a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels. It is added to the DNA sample before loading to enable visualization of the fragments within the gel or can be added in the electrophoresis buffer. The binding of ethidium bromide to DNA alters its mass and rigidity, and therefore its mobility. 

What is phage virus ?

What is phage ?

PHAGE can also replicate via the Lysogenic cycle. The phage genome is integrated into the host chromosome and is inherited into the chromosomes of all daughter bacteria. This "prophage" can be induced to enter the lytic cycle and kill its host by a variety of stresses like UV light

What is the Bacteriophage?

What is Bacteriophage ?

Bacteriophages replicate via the lytic phase cycle and the phage genome is injected into the cell, phage genes are expressed and phage proteins and DNA are made, progeny phage are packaged, and the cell is lysed. Two genetically different phage that infect the same host cell may recombine during the lytic cycle

What is Recombinant DNA Technology in brief

What is a Recombinant DNA ?

DNA molecules constructed outside the living cells that is in vitro by joining natural or synthetic DNA segments that can replicate in a living cell.

What is the Goals of Recombinant DNA Technology?

a) To isolate and characterize a gene
b) To make desired alterations in one or more isolated genes
c) To return altered genes to living cells

What is Basic Tools of Recombinant DNA Technology?

Nucleic Acid Enzymes DNA and RNA polymerases, reverse transcriptase, DNA ligases, Restriction endonucleases and many more.

How to isolate a plasmid from a bacteria ?

How to isolate a plasmid from a bacteria ?

Plasmids are easily isolated from bacterial cells Plasmid isolation takes advantage of the unique structural properties of plasmids. Plasmids are small,super coiled circular pieces of Dana.Unlike the much larger bacterial chromosomes(which is also circular),plasmids are quite resistant to permanent den. Today,most laboratories use commercial kits for plea mid isolations,because the kits are convenient and relatively inexpensive.the kits give good yields of highway a little,while avoiding the need for organic denaturant ants.A variety of less expensive,but somewhat more time-consuming,procedure have been described for investigators who want to make their own reagents.These procedures generally give good yields of death at is slightly less pure in a purified with the kits.Whatever the isolation procedure,the general principles of plasmid isolation are the same.The figure and paragraphs below summarise regs general principles used for plasmid isolation.

1. Lysis and denaturation-

Strong denaturating conditions to weaken the tough bacterial drop wall.The most common procedures use a combination of strong base and a detergent.The detergents help to solubility lipids in the,allowing the denaturants to enter the cell. Proteins,because of their fragile structures,are irreversibly denatured.The treatment also breaks the hydrogen bonds holding together the chromosomal and plasmid DNA.

 2. Neutralization-

Neutralization allows complementary DNA strand store anneal and causes proteins to precipitate.Plasmids renature because they have super coiled structures that have held the two strands of the helix together denaturation.chromosomal DNA a is not able to renature,however,because it's longer strands have become mixed with denatured proteins. Samples must be mixed gently at this step to prevent fragmentation of the long, chromosomal DNA into pieces that might be able to reanneal and co-purify with the plasmids.  

3. centrifugal- 

on plasmid Dana is separated from large aggregates of precipitated protea chromosomal in a by centrifugal ion. 

4. Additional purification -

Plasmids are further purified by organic extraction or adsorption in resin.

What is plasmid DNA?

What is plasmid DNA?

=>

Plasmid is a double standed ,self replicating,extra chromosomal DNA Plasmids are cloning vectors that are widely used in molecularbiology andthey play an important roles in the laboratory.Plasmids are small, circular pieces of DNA  that replicate independently of the host chromosome.The first plasmids used in the lab were derivatives of naturally I occurring plasmids found in vacate.Since their discovery,molecularbiologists have added many features to plasmids to suit a variety of applications.In this lab,each team will I so late three plasmids from bacterial strains.

Plasmid DNA are red circular one

a short notes on Insulin of human body

                                       Human Insulin 

Human insulin is a globular protein with a molecular weight of about 5,800 kd, consisting of 51 aminoacid residues organised in two polypeptide chains (A and B), linked by two disulphide bonds. Chain A consists of 21 residues with an extra disulphide bond between A6 and A11; chain B consists of 30 aminoacids. Complete synthesis of the human insulin molecule was achieved in 196610. Insulin exists as a monomer only at low concentrations while it shows propensity to aggregate into stable dimers at higher concentrations, in aqueous solution at pH 2-8 and into hexamers in the presence of zinc ions. The hexamer, in which chain A constitutes much of the polar surface, is almost spherical in structure, with a diameter of 5 nm and a height of 3.5 nm. Polymerisation of the hormone has major pharmacological implications.

What Is Cancer?

 What Is Cancer?

=> Cancer results from a series of molecular events that fundamentally alter the normal properties of cells. In cancer cells the normal control systems that prevent cell overgrowth and the invasion of other tissues are disabled. These altered cells divide and grow in the presence of signals that normally inhibit cell growth; therefore, they no longer require special signals to induce cell growth and division. As these cells grow they develop new characteristics, including changes in cell structure, decreased cell adhesion, and production of new enzymes. These heritable changes allow the cell and its progeny to divide and grow, even in the presence of normal cells that typically inhibit the growth of nearby cells. Such changes allow the cancer cells to spread and invade other tissues. The abnormalities in cancer cells usually result from mutations in protein-encoding genes that regulate cell division. Over time more genes become mutated. This is often because the genes that make the proteins that normally repair DNA damage are themselves not functioning normally because they are also mutated. Consequently, mutations begin to increase in the cell, causing further abnormalities in that cell and the daughter cells. Some of these mutated cells die, but other alterations may give the abnormal cell a selective advantage that allows it to multiply much more rapidly than the normal cells. This enhanced growth describes most cancer cells, which have gained functions repressed in the normal, healthy cells. As long as these cells remain in their original location, they are considered benign; if they become invasive, they are considered malignant. Cancer cells in malignant tumors can often metastasize, sending cancer cells to distant sites in the body where new tumors may form.